The main goal of a mitotic cell is to equally seggregate its chromosomes and centrosomes between two daughter cells. The careful orchestration of cytoskeletal and chromosomal events requires coordinated action by members of the CDK (cyclin-dependent kinase), Plk (polo-like kinase) and Aurora kinase families. The study of these kinases, their regulatory subunits and substrates has attracted considerable attention in recent years, in part because they are all candidate targets for cancer therapy. Indeed, during mitosis, a spectacular reorganization of the cytoskeleton occurs that builds a bipolar microtubule spindle that assures proper segregation of chromosomes and requires a number of precisely coordinated cell-cycle events to occur. By the end of S-phase, the cell must have duplicated its centrosome and replicated its DNA. At the end of prophase, the duplicated and matured centrosomes must have become separated. During prometaphase, the two centrosomes and the chromosomes nucleate highly dynamic mitotic microtubules that assemble a bipolar spindle. During progression from prometaphase to metaphase, the chromosomes must become bi-orientated and aligned at the metaphase plate. Bi-orientation is achieved by microtubule-organized attachment of kinetochore pairs to opposite centrosomes. During this process, the mitotic checkpoint is continuously activated; it controls microtubule attachment to the kinetochores and tension. When these two conditions are satisfied, the checkpoint signals are switched off, the chromatids separate and anaphase proceeds. In telophase, nuclear division occurs and the cell undergoes cytokinesis. Finally, each daughter cell receives one set of chromosomes and one centrosome.
Considering the complexity of mitosis, not surprisingly there are many mitotic defects that can lead to the formation of aneuploid daughter cells, i.e. cells that possess an altered content of DNA (abnormal number of chromosomes). To prevent the appearance of such aneuploid cells, the cell will enter into mitotic catastrophe, i.e. a type of cell death as a result of DNA damage or deranged spindle formation coupled to the debilitation of different checkpoint mechanisms that would normally arrest progression into mitosis and hence suppress catastrophic events until repair has been achieved. Cells that fail to execute mitotic catastrophe in response to mitotic failure are likely to divide asymmetrically, with the consequent generation of aneuploid cells.
Most tumors develop in an (oligo) clonal and stochastic manner, through a multi-step process. It is accordingly a hypothesis that one of the mechanisms that contribute to oncogenesis consists of ‘cytogenetic catastrophe’, i.e. the failure to activate mitotic catastrophe in response to mitotic failure (Castedo, M., et al., Oncogene (2004) 23, 2825-2837). In these circumstances aneuploidization could result from the asymmetric division of polyploid cells, generated from an illicit cell fusion, as it may occur in vivo or from endoreplication/endomitosis. Indeed, polyploidy is frequently observed in neoplasia and constitutes a negative prognostic factor, while aneuploidy is a near to general characteristic of cancer.
As already mentioned above, the networks of kinases that regulate the mitotic events are all candidate targets for cancer therapy. For example, Aurora A is an oncogenic serine/threonine kinase that plays a role in centrosome separation and in the formation of the mitotic bipolar spindle. Aurora B is required for chromosome alignment, kinetochore-microtubule bi-orientation, activation of the spindle assembly checkpoint and cytokinesis. Both Aurora A and B are upregulated in various cancers, Aurora A is commonly amplified in melanoma and cancers of the breast, colon, pancreas, ovaries, bladder, liver and stomach. Aurora B is frequently increased in tumors such as colorectal cancer and high-grade gliomas, and Aurora B overexpression in CHO cells results in an increased invasiveness, suggesting a role for Aurora B in tumorigenesis (Carvajal, R. D. et al., Clin. Cancer Res. (2006) 12(23), 6869-6875).
Another member of the kinases involved in cellular mitosis, are the cyclin-dependent kinases CDKs. The family of cyclin-dependent kinases lies at the core of the machinery that drives the cell division. It is for example, well established that CDK1, formerly called Cdc2, interacts with its obligate allosteric activator, cyclin B1 to form an active heterodimer, the ‘mitosis-promoting factor’. The mitosis-promoting factor induces mitosis by phosphorylating and activating enzymes regulating chromatin condensation, nuclear membrane breakdown, mitosis-specific microtubule reorganization and actin cytoskeleton allowing for mitotic rounding up of the cell. Aberrant mitotic entry, for example before the completion of DNA replication, can result in cytogenic catastrophe as observed in many tumor cells. Obviously, this requires the activation of CDK1, and it is currently assumed that premature entry of active CDK1/cyclin B1 complex into the nucleus suffices to cause premature chromatin condensation that may result in aneuploidization (Castedo M. et al., supra). This increasing body of evidence provides a link between tumor development and CDK related malfunctions and led to an intense search for inhibitors of the CDK family as an approach to cancer therapy.
Final members of the kinases involved in cellular mitosis are Polo-like kinases (PLKs). PLKs are key enzymes that control mitotic entry of proliferating cells and regulate many aspects of mitosis necessary for successful cytokinesis, including centrosome duplication and maturation; DNA damage checkpoint activation; bipolar spindle formation; Golgi fragmentation and assembly; and chromosome segregation (Barr, F. A. et al., Nat. Rev. Mol. Cell. Biol. 2004, 5, 429-441). Given the established role of PLKs as mitotic regulators, they have been regarded as validated mitotic cancer targets for a number of years. In addition, recent studies demonstrate that changes of intracellular levels of PLKs are involved in the control of cell growth. For example, PLK1 when fused to an antennapedia peptide and efficiently internalized into cells caused an inhibition of cancer cell proliferation (Yuan, J., et al., Cancer Res. 62, 2002, 4186-4190), whereas downregulation of PLK1 by antisense induced the growth inhibition of cancer cells (Spankuch-Schmitt, B., et al., Oncogene 21, 2002, 3162-3171). PLK2 was recently found to be a novel p53 target gene and RNAi silencing of PLK2 leads to mitotic catastrophe in taxol-exposed cells (Burns, T F., et al., Mol Cell Biol. 23, 2003, 5556-5571). For PLK3 it was found that it induces cell cycle arrest and apoptosis through perturbation of microtubule structure (Wang, Q., et al., Mol Cell Biol. 22, 2002, 3450-3459) and PLK4 was shown to be transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Li, J., et al., Neoplasia 7, 2005, 312-323). Thus confirming that targeting PLKs with conventional small-molecule agents may be a valid and effective anticancer strategy with potential to synergize with established DNA-damage and antimitotic chemotherapies. PLK4 was also found to be required for centriole duplication and flagella development. The absence of centrioles, and hence basal bodies, compromises the meiotic divisions and the formation of sperm axonemes. This implies a possible use of PLK4 antagonists as male contraceptives.
We have now found that, certain indolin-2-ones and aza-indolin-2-ones possess potent anti-tumor activity. Without wishing to imply that the compounds disclosed in the present invention possess pharmacological activity only by virtue of an effect on a single biological process, it is believed that the compounds provide an anti-tumor effect by way of inhibition of one or more of protein kinases that are involved in the regulation of cellular mitosis and which lead to cytogenetic catastrophe in case of aberrant activity.
The compounds of the present invention, were also found to have Glycogen synthase kinase-3 (GSK-3) inhibitory activity and are accordingly useful in the prevention or treatment of diseases mediated through GSK-3 activity such as bipolar disorder (in particular manic depression), diabetes, Alzheimer's disease, leukopenia, FTDP-17 (Fronto-temporal dementia associated with Parkinson's disease), cortico-basal degeneration, progressive supranuclear palsy, multiple system atrophy, Pick's disease, Niemann Pick's disease type C, Dementia Pugilistica, dementia with tangles only, dementia with tangles and calcification, Downs syndrome, myotonic dystrophy, Parkinsonism-dementia complex of Guam, aids related dementia, Postencephalic Parkinsonism, prion diseases with tangles, subacute sclerosing panencephalitis, frontal lobe degeneration (FLD), argyrophilic grains disease, subacutesclerotizing panencephalitis (SSPE) (late complication of viral infections in the central nervous system), inflammatory diseases, depression, cancer, dermatological disorders such as baldness, neuroprotection, schizophrenia, pain, in particular neuropathic pain. GSK3 inhibitors can also be used to inhibit sperm motility and can therefore be used as male contraceptives. Therefore, the invention also provides the use of the macrocyclic indolin-2-ones and aza-indolin-2-ones as male contraceptives.
In particular, the compounds of the present invention are useful in the prevention or treatment of Alzheimer's disease; diabetes, in particular type 2 diabetes (non insulin dependent diabetes); bipolar disorder; cancer including lung cancer (especially non small-cell lung cancer), breast cancer, liver cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer such as colon, bladder, rectal or stomach cancer and papillary carcinomas (such as papillary thyroid cancer) as well as in squamous cell cancers of the head and neck and in oesophageal cancers including oropharyngeal cancer; pain, in particular neuropathic pain; depression; inflammatory diseases including allergies and asthma, MS, RA, arteriosclerosis, arthritis or IBD.